The flawless execution of cell division is essential to the generation and survival of all organisms. During every cell cycle, chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of all tumors and many birth defects. Our goal is to elucidate the mechanisms that ensure accurate chromosome segregation and therefore contribute to understanding the basis of human disease. We are studying chromosome segregation in budding yeast because it is amenable to both genetic and biochemical analyses and the mechanism of chromosome segregation is fundamentally conserved. Chromosome segregation requires that the kinetochores of duplicated chromosomes (sister chromatids) biorient such that they bind to microtubule (MTs) arising from opposite spindle poles. Although it is essential that every pair of sister kinetochores make bioriented MT-kinetochore attachments, the mechanisms that establish, maintain, and correct errors in biorientation are still largely unknown. The only protein known to be essential for kinetochore biorientation is the conserved IpH (Aurora B) protein kinase whose localization and activity is regulated by the SIM 5 (INCENP) protein and opposed by Glc7, the catalytic subunit of protein phosphatase I. We have identified additional genes that likely regulate biorientation because they become essential when IpM function is impaired. We will therefore characterize the role of these genes in biorientation and identify the mechanisms that they use to regulate biorientation. Although IpH and Glc7 are critical for kinetochore biorientation, few molecular targets of these enzymes have been identified. We have therefore developed a method to purify kinetochores that will allow us to identify post-translational modifications associated with these enzymes and biorientation. Finally, we will take complementary genetic and biochemical approaches to understand how the activities of IpH and Glc7 are spatially and temporally coordinated to ensure that biorientation defects are detected and corrected. Taken together, these studies should lead to a better understanding of chromosome segregation and the maintenance of genomic stability in all eukaryotes.